Atherosclerosis is the major cause of death and disability in Westernized societies and is rapidly becoming so in developing nations. Despite significant medical progress our knowledge is incomplete and we still need to discover new genes and pathways in atherogenesis. Mouse models of atherosclerosis provide a complementary approach to human studies for such discoveries, and in an intercross between C57BL/6J and FVB/N mice on the apoEKO background we identified an atherosclerosis susceptibility locus on proximal mouse chromosome 10 (Ath11). Our work is at the forefront of the application of Quantitative Trait Locus (QTL) analysis not just to atherosclerosis but to all complex traits. We showed this locus contains 2 regions, a distal 10b region of 5 genes with Raet1e as a strong candidate gene, and a proximal 10a region of 21 genes with Esr1 an appealing candidate. The overall objective of this grant request is to confirm Raet1e and Esr1 as the culprit genes and elucidate their mechanisms of action. Raet1e is an MHC class I-like molecule expressed on the surface of stressed cells. It is a ligand for NKG2D, a receptor on the surface of NK, NKT, and T-Reg cells and some macrophages. This interaction plays a key role in the elimination of tumors and infected or damaged cells by the innate immune system. Our studies suggest aortic over expression of Raet1e is atheroprotective. To confirm this, we have made BAC Raet1e transgenic mouse strains and by breeding the transgene onto the apoEKO background we hope to prove our hypothesis. We will then identify the sequence variations between C57 and FVB in the Raet1e gene in the region of interest and find out which sequence variation is responsible. To determine how Raet1e over expression is atheroprotective we will analyze aortic gene expression patterns and cytokine profiles of mice with and without the Raet1e transgene. Using apoEKO and apoEKO NKG2DKO mice we will resolve if the effect of over expression of Raet1e on atherosclerosis is dependent or independent of the NKG2D receptor. We will identify by FACS analysis of splenic and aortic cells the immune cells involved in this process and with functional studies, such as cytotoxicity and proliferation assays, determine how over expression of Raet1e alters the physiological state of these cells. Esr1 is the main nuclear hormone receptor for estrogens. We will confirm Esr1 as the 10a region gene by expression analysis in subcongenic strains as well as comparing C57 and FVB Esr1 gene sequence, aortic mRNA and protein levels, and ESR1 function to identify the molecular and mechanistic basis of Esr1 as an atherosclerosis modifier gene. If we do not find support for Esr1 as a candidate gene for the 10a region we will identify, among the other 20 genes in the 10a region, the responsible gene by characterizing new subcongenic strains that further narrow the 10a region, and evaluate the most promising candidate genes by gene sequencing, gene expression analysis, and functional studies.